Figure 5.

Inhibitory effects of PD-98059 on gonadotropin-releasing hormone agonist (GnRHa)-induced 3β-hydroxysteroid dehydrogenase (3β-HSD) expression. Leydig cells were pretreated with PD-98059 (50 µmol l⁻¹) for 20 min and then stimulated with 100 nmol l⁻¹ GnRHa for 24 h. Cells were harvested, and total RNA and protein were extracted. 3β-HSD expression was analysed by real-time PCR (a) and Western blot analysis (b). 3β-HSD mRNA was normalized as a ratio to the amount of Rp119 mRNA. The intensities of the protein bands in the western blot were quantified, and 3β-HSD protein levels were normalized to that of β-actin. Data are presented as mean±s.e.m. of three separate experiments. *, P < 0.05, compared with GnRHa; #, P < 0.05, compared with the control.